Simaltaneous Determination Of Melphalan And Its Process .
IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS)e-ISSN:2278-3008, p-ISSN:2319-7676. Volume 12, Issue 5 Ver. I (Sep. – Oct. 2017), PP 69-78www.iosrjournals.orgSimaltaneous Determination of Melphalan and Its ProcessRelated Impurities Using a Stability-Indicating and ValidatedReverse Phase Hplc Method in a Short Run Time.JagadeswaraRao K1,2, Murali Mohan SV 2and *Rama Rao Malla11Department of Biochemistry, Cancer Biology Lab, GIS, GITAM University,Visakhapatnam, India2Analytical Research and development Department, Shilpa Medicare Limited, India* Corresponding author: Prof. Rama Rao MallaAbstract: Degradation pathway of melphalan was validated by stability indicating reverse phase liquidchromatographic method. melphalan was subjected to stress condition using acid, base or by oxidation, andphotolysis. Significant degradation was observed in acid and base stress conditions. The stress samples wereassayed against a qualified reference standard and the mass balance was found close to 98.2 %. Efficientchromatographic separation was achieved on a Luna C18 (4.6 x 150 mm) 3 μm stationary phase with simplemobile phase combination. In the developed LC method, the resolution of melphalan and potential impuritiessuch as Dimer impurity, monohydroxy impurity, phthalimide, ethyl ester, elongated melphalan, dihydroxy andmonolakylate was found to be more than 2.0. Regression analysis showed correlation coefficient (r) of greaterthan 0.999 for melphalan and eight potential impurities. This method was capable of detecting the impurities ofmelphalan at a level of 0.02 % with respect to test concentration of 0.5 mg/mL. The developed rapid LC methodwas showed specificity, linearity, accuracy, precision and robustness for impurities.Key words: Melphalan, method development, validation, precision and ------------------------------------ ---------Date of Submission: 25-08-2017Date of acceptance: ----------------------------------- ----------I.IntroductionMethod development is a multi step process involves adoption of existing method, making minorchanges suitable for the novel application and developing a method for the estimation of drug using a HPLC.Method validation is a process normally followed to acceptability of a analytical techniques used to determinedrugs in pharmaceutical dosages.Melphalan is chemically known as 4-[bis (2-chloroethyl) amino]-L-phenylalanine [1-3]. It is Lphenylalanine derivative of nitrogen mustard (L-PAM). It is a bifunctional alkylating agent, active against ofmultiple myeloma, lymphomas, malignant melanoma and ovarian carcinoma [4-5]. The molecular formula isC13H18Cl2N2O2 and the molecular weight is 305.20. The structural formula of melphalan was depicted in Figure1 [6]. Active melphalan is a L-isomer and was first synthesized in 1953 by Bergel and Stock [7]. The D-isomer isless active and requires high dosage to produce effects on chromosomes. The racemic (DL-) form is known assarcolysin [8]. Melphalan is normally insoluble but melphaln hydrochloride is soluble in water and has a pKa1 of 2.5. The injection form of melphalan hydrochloride was supplied as a sterile, non pyrogenic, freeze-driedpowder. Each single-use vial contains melphalan hydrochloride equivalent to 50 mg melphalan and 20 mgpovidone. It is reconstituted using the sterile diluents. Sodium citrate 0.2 g, propylene glycol 6.0 mL, ethanol(96%) 0.52 mL, and water in a total of 10 mL [9]. Melphalan hydrochloride injection is administeredintravenously. Some of the chromatographic methods and Moss spectroscopy methods were reported for theestimation of melphalan in tablets dosage forms. Melphalan can be determined by different spectrophotometricmethods [10-19]The present study describes a simple, convenient efficient and robust HPLC method for thesimultaneous estimation of melphalan content and related impurities in melphalan drugs.II.Experimental2.1 Chemicals:The melphalan and its related impurities such as Dimer impurity, monohydroxy impurity, phthalimide,ethyl ester, elongated melphalan, dihydroxy and monolakylate used in the present study were of 99% purityand provided by PS3 LABORATORIES LIMITED LLP, Hyderabad. Acetonitrile, trifloro acetic and methanolof HPLC grade were purchased from Merck (Darmstadt, Germany).All other chemicals used in the present wereanalytical grade were obtained from commercial source.DOI: 10.9790/3008-1205016978www.iosrjournals.org69 Page
Simaltaneous Determination Of Melphalan And Its Process-Related Impurities Using A Stability2.2 Equipment’s:The Liquid chromatography system was used for the method development and method validation. Thesystem consists of binary separation module equipped with an auto sampler and a photo diode array detector(Waters, USA). The output signal was monitored and processed using Empower software (Empower 3) viewsonic computer. Photo stability and thermal stability studies were carried out using photostability chamber(Newtronic life care, India). Dry hot air oven (Newtronic life care, India), respectively.2.3 Chromatographic conditions:The chromatographic column, Luna C18 (4.6 x 150 mm, 3 μm) was used with a mobile phase-Acontaining 750 mL of water, 250 mL of acetonitrile and 1 mL of trifloro acetic and mobile phase-B containing250 mL of water, 750 mL of acetonitrile and 1 mL of trifloro acetic. The flow rate was fixed at 1.0 mL/min, asautosampler, column temperatures were maintained at 25 and 5 C respectively and detection wavelength wasset at 265 nm. 10µL of sample methanol was injected into system. Total system run was carried out for 40 min.The gradient program was fixed as shown in the table 1.2.4 Preparation of standard solutions and sample solutions:The system suitability test solution (0.5 mg/ml) was prepared by suspending melphalan standard inmethanol using ultrasonic bath. The quantification test solution was prepared by diluting system suitability testsolution with methanol (1:100). The limit of quantification test solution (0.05%) was prepared by dilutingquantification test solution to with methanol (1:20). Unless otherwise stated freshly prepared solution was used.The commercially obtained melphalan sample was reconstituted with 10 mL water and diluted with methanol (5mg/mL). The sample solution was transferred to 10 mL sample solvent and filtered through a 0.45µm nylon diskmembrane filter.III.Method Development3.1 Method development and optimizationMethod development was initiated by multiple experiments. In experimental setup-1 mobile phase- Aconsisting of 950 mL of water, 50 mL of acetonitrile and 1 mL of trifloroacetic acid and mobile phase-Bconsisting of 50 mL of water, 950 mL of acetonitrile and 1 mL of trifloroacetic acid, stationary phase (WatersC18 150 x 4.6 mm, 5µm) detection wavelength 262 nm and column oven temperature 25 C were used. TheInjection volume 10 µL of impurity mixture, was analyzed. In experimental setup-2. Luna C18 (4.6 x 150 mm, 3μm) column was used with a mobile phase-A containing of 950 mL of water, 50 mL of acetonitrile and 1 mL oftrifloroacetic acid and mobile phase -B consisting of 50 mL of water, 950 mL of acetonitrile and 1 mL oftrifloroacetic acid were used with the flow rate of 1.0 mL/min, column temperature of 30 C, auto samplertemperature of 5 C and detection wavelength of 262 nm. In experimental setup- 3, same mobile phase A and B,stationary phase , flow rate of 1.0 mL/min, detection wavelength of 260 nm and column oven temperature of25 C were used for separation of melphalan related impurities.3.2 Method validation parameters:3.2.1 Stress studies / Specificity:The stress studies/specificity was used to evaluate the ability of the method to resolve possiblesubstance, degradation products of melphalan and its other impurities. For validation of method impuritysamples and degradation products (5 PPM) were spiked in the melphalan sample. Forced degradation studieswere performed on melphalan to infer stability-indicating property and specificity of validating method. Thestress conditions used the degradation study were light (ICH Q1B), heat (100 C), acid (1N Hydrochloric acid),base (0.01 M sodium hydroxide) and oxidation (30% Hydrogen peroxide). The samples were exposed for 24 h,to heat and light studies where as samples were treated for 8 h with acid and base hydrolysis and also foroxidation studies. The peak purity of the melphalan stressed samples were validated using photo diode arraydetector (water’s).The purity angle was set within the purity threshold limit for all of the stressed samples andcontents of impurities were calculated for the stress samples against a qualified reference standard. The massbalance (% assay % of impurities % of degradation products) was calculated for all of the samples.3.2.2 Method validation:The developed method was validated as per ICH recommendations. The system precision wasinvestigated by injecting six individual preparations (5µg/ml) of melphalan spiked with specification level ofeach of Dimer impurity, monohydroxy impurity, phthalimide, ethyl ester, elongated melphalan, dihydroxy andmonolakylate. The %RSD of the areas of above mentioned melphalan impurities was calculated. Theintermediate precision of method was confirmed by different analysts and different instruments. The precisionDOI: 10.9790/3008-1205016978www.iosrjournals.org70 Page
Simaltaneous Determination Of Melphalan And Its Process-Related Impurities Using A Stabilityof the method was further evaluated by the analysis of six independent analysis of a test samples of melphalanagainst a qualified reference standard. The %RSD of six independent test values was calculated.3.2.3 Limit of detection (LOD) and limit of quantification (LOQ):The LOD and LOQ of Dimer impurity, monohydroxy impurity, phthalimide, ethyl ester, elongatedmelphalan, dihydroxy and monolakylate and melphalan were evaluated at 3:1 and 10:1 signal-to noise ratio byinjecting a series of dilute solutions with known concentrations. The precision study was also performed at theLOQ level by injecting six individual samples and calculated the %RSD of the areas.3.2.4 Linearity:Linearity test solutions were prepared by diluting stock solution from 50 to 150% of the analyte for therelated substance method. The peak area versus concentration details were analyzed with least-squares linearregression. The linearity test solutions were prepared by diluting the impurity stock solution to the requiredconcentrations for the related substance method. The solutions were diluted at six different concentration levelsfrom the LOQ to 150%.The slope and y-intercept of the calibration curve was observed. The peak area versusconcentration data was analyzed using least squares linear regression. The linearity test solutions were preparedby diluting the impurity stock solution to the required concentrations for the related substance method. Theslope and y-intercept of the calibration curve were reported.3.2.5 Accuracy:The accuracy of the related substance method was evaluated in triplicate at three concentration levels,50, 100 and 150% and the percent recovery was calculated. The impurities, Dimer impurity, monohydroxyimpurity, phthalimide, ethyl ester, elongated melphalan, dihydroxy and monolakylate were spiked intomelphalan and recovery experiments were performed to determine the accuracy of the related substance methodfor quantification of impurities. The study was carried out in triplicate at 0.5, 1.0 and 1.5 % of the analyteconcentration (0.5 mg/ml). The percent recovery of above mentioned impurities was calculated.3.2.6 Robustness:The robustness of developed method of evaluated at different experimental conditions such as flow rateand column temperature. The flow rate of the mobile phase was maintained 1.0 ml/min. To study the effect ofthe flow rate on the resolution, the flow rate was changed every time by 0.1 units from 0.9 and 1.1 ml/min. Theeffect of column temperature on the resolution was studied from 20 C -30 C. The detector wavelength wasmaintained 260 nm. To study the effect of the wavelengths on the resolution, the wavelength was changed to258 and 262 nm. To study the effect of the organic solvent ratio on the resolution, the organic solvent ratio waschanged 2.0%. In all these varied conditions, the components of the mobile phase was remain same.3.2.7 Solution stability and mobile phase stability:The stability of melphalan solution in the proposed method was tested out by placing the sample andreference standard solutions in a tightly capped volumetric flasks at room temperature for 8 h. The samplesolution was assayed at 8 h interval during the study period. The mobile phase stability was evaluated byassaying the freshly prepared sample solution against freshly prepared reference standard solution at 6 hinterval. The prepared mobile phase maintained same during the study period. The %RSD of the melphalanimpurities was calculated for the mobile phase as well as solution stability experiments. The amount of Dimerimpurity, monohydroxy impurity, phthalimide, ethyl ester, elongated melphalan, dihydroxy and monolakylatewas determined at 6 h intervals up to the study period. The stability of mobile phase was evaluated for 72 h byinjecting the freshly prepared sample solution at every 8 h interval. The content of melphalan and its impuritiesDimer impurity, monohydroxy impurity, phthalimide, ethyl ester, elongated melphalan, dihydroxy andmonolakylate were determined in the test solution. The prepared mobile phase was maintained remain sameduring the study period.IV.Results And Discussion4.1 Method development and optimizationThe information of active pharmaceutical ingredient melphalan and official product methods were notavailable in USP, BP and EP pharmacopeia. According to API route of synthesis, there were seven specifiedimpurities in melphalan drug such as Dimer impurity, monohydroxy impurity, phthalimide, ethyl ester,elongated melphalan, dihydroxy and monolakylate. The current study was aimed to separate melphalan fromimpurities and degradants in a shorter run time using a RP-HPLC stability indicative method. The injectionsequence of related substance and assay method of melphalan was depicted in the table 1. The results ofexperimental setup 1 of method development study reveals that the impurity peaks not separated and peakDOI: 10.9790/3008-1205016978www.iosrjournals.org71 Page
Simaltaneous Determination Of Melphalan And Its Process-Related Impurities Using A Stabilityshapes were not satisfactory. The results of experimental setup-2 was also not satisfactory for impurity peakseparation and peak shapes. However, in experimental setup-3 separation of impurity peaks with sharp peakshapes was achived. These results indicate that Luna (C18 4.6 x 150 mm, 3 μm) sample cooler temperature 5 Cand column oven temperature was 25 C found to be ideal for separation and sharp peak shapes.Optimization of mobile phase was performed based on resolution of the drug, asymmetric factor andtheoretical plates obtained for melphalan. The results indicate that mobile phase A consisting of 950 mL ofwater, 50 mL of acetonitrile and 1 mL of trifloroacetic acid and mobile phase B consisting of 50 mL of water,950 mL of acetonitrile and 1 mL of trifloroacetic acid, a mobile phase flow rate of 1.0 ml/min was found to besatisfactory, achieved good separation and showed symmetric peak for melphalan and related impurities (Table2). The results reveals that column oven temperature of 25 C, sample cooler temperature of 5 C. Retention timeof 15 min. and detection wavelength at 260 nm were found to be ideal parameter for complete resolution of thepeaks with clear baseline separation in the estimation of melphalan using reverse phase HPLC.Table 1. HPLC conditions for gradient separation melphalan impurities.Time (min)01528323636.140Flow (mL/min)1.01.01.01.01.01.01.0Mobile phase A (%)956530009595Mobile phase B (%)53570100100554.0.1 Stress studies / Specificity:The stressed samples of melphalan were subjected to forced degradation studies using light and heat.Significant degradation of melphalan and its products was detected with thermal, acid and oxidation, which maylead to formation of unknown degradation products with RRT of 1.19 (Table 3).Table 3. Retention time of melphalan and its impurities and degradantsS.No.123Name of the impurityMonohydroxyDimerMelphalanRetention time in (min)7.117.716.2RRT0.431.091.00The peak purity test using PDA detector confirmed that the melphalan peak and the degraded productspeaks were homogeneously pure in all the analyzed stress samples. The degradation studies against a qualifiedreference standard of melphalan reveals that the mass balance of the stressed samples was close to 99.5%. Theseresults further indicate that the developed method for estimation of melphalan was not affected by the presenceof melphalan and monohydroxy, Dimer and its degradation products, confirming the stability-indicating power(Table 4).Table 4. Effect of different stress conditions on a resolution melphalan and its impurities by newlydeveloped HPLC methodML: Melphalan, MM: Monohydroxy MD: Dimer, MP: Phthalimido, MEE: Ethyl esterME: Elongated, MM: Monolakylate, MDY: DihydroxyDOI: 10.9790/3008-1205016978www.iosrjournals.org72 Page
Simaltaneous Determination Of Melphalan And Its Process-Related Impurities Using A Stability4.0.2 System suitability and System precision:The melphalan limit of quantification solution and system suitability solution were injected into the respectivechromatographic condition and recorded the melphalan peak areas. The observed signal to noise was found to be20.6 and % RSD for six replicate injections was observed as 3.0% (Table 5).4.0.3 Method precision:The results of method precision study of six individual sample solutions within 10.0 % RSD show that % RSDof unknown impurity and total impurities were 9.8 and 7.0, respectively (Table 6).Table 6. Method precision for separation of melphalan and its impurities by developed HPLC e0.040.07STDEV0.000.01%RSD9.87.04.0.4 Intermediate precision:The method precision study of six individual sample solutions within 10.0 % RSD show that % RSD ofunknown impurity and total impurities were 8.4 and 7.0, respectively (Table 7).Table 7. Intermediate precision for separation of melphalan and its ityTotal 50.07STDEV0.000.01%RSD8.47.0ML: Melphalan, MM: Monohydroxy MD: Dimer, MP: Phthalimido, MEE: Ethyl ester ME: Elongated, MM:Monolakylate, MDY: Dihydroxy4.0.5 Accuracy:The percent recovery of melphalan was ranged from 0.03 to 0.15 at the 100% level of unknown impurities in themelphalan drug Figure 3. The results on the accuracy of method was depicted in the (Table 8).the resultsindicate that the recovery at LOQ, 50,100 and 150% was 110,109,95.6 and 95.6 respectively indicating accuracyof developed method.Table 8. Data derived from of melphalan by proposed HPLC methodS.No.123123DOI: 40.030.050.060.05www.iosrjournals.org73 Page
Simaltaneous Determination Of Melphalan And Its Process-Related Impurities Using A rageSTDEV%RSDAverageSTDEV%RSDRecovery at LOQ %Recovery at 50 %Recovery at 100 %Recovery at 150 50%LOQ50%100%150%4.0.6 Linearity:The linear calibration plot of the method was obtained in the tested calibration range of 50-150% leveland the correlation coefficient obtained was 0.999. These results indicate significant correlation between thepeak areas and analyte concentration. The linear calibration plot for the related substance was determined in thecalibration range for melphalan was 150% with respective to LOQ, a correlation coefficient of greater than0.999 was obtained. In this range, the linearity was checked for the related substance at same concentrationrange for three consecutive days. The % RSD values of the slope and y-intercept of the calibration curves wereachieved within 10%. These results showed significant correlation between the peak areas and concentrations ofmelphalan. The residuals were within 10% scattered with respect to 100% concentration response. Thesensitivities were scattered within 10% with respect to 100% concentration sensitivity (Table 9).Table 9. Regression and precision data analysis of melphalan by developed HPLC methodName of the ImpurityLOD %LOQ %Slope (m)Intercept (C)Correlation coefficientPrecision (%RSD)Melphalan0.004%0.0121084516.730.99995.24.0.7 Limit of quantification (LOQ) precision:The LOQ precision study was carried out within 10.0 % RSD. The results show that % RSD of melphalan were2.01 (Table 10).4.0.8 Robustness:At deliberately, modified chromatographic conditions such as flow rate, pH, solvent ratio and columntemperature etc., the resolution between the closely eluting impurities, Dimer impurity, monohydroxy impurity,phthalimide, ethyl ester, elongated melphalan, dihydroxy and monolakylate resolution was greater than 2.0. Thevariability of both melphalan and the impurities area response was within 5% emphasizing the robustness ofthe developed HPLC method.4.0.9 Solution stability and Mobile phase stability:The results of solution stability and mobile phase stability indicate the % RSD of the related substancesmethod of melphalan was less than 1%. These results indicate that no significant changes in the content ofmelphalan during the solution and mobile phase stability experiments The results of the solution and mobilephase stability experiments confirm that the sample solutions and mobile phase used during the relatedsubstance determinations were stable up to 96 h. The mobile phase was proven to be proved stable upto oneweek days.DOI: 10.9790/3008-1205016978www.iosrjournals.org74 Page
Simaltaneous Determination Of Melphalan And Its Process-Related Impurities Using A StabilityV.ConclusionThe degradation pathway of melphalan was established as per ICH recommendations. The gradient LCmethod was developed and used for stress studies and also for quantification impurities of melphalan drug. Thebehavior of melphalan under various stress conditions was studied. The thermal and all the degradation productsand processed impurities were well separated from the melphalan and related impurities demonstrates thestability-indicating power of the method. This method is sensitive and can detected up to 0.02% impurities. Thismethod was also precise, accurate and stability indicative. Within the recommendations of ICH. The developedmethod can be used to determine the impurities in melphalan injection in the routine and stability sampleanalysis.AcknowledgementsThe authors wish to thank the management of Terra Scientific Pvt. Ltd, Hyderabad and India for supporting thiswork. We thank management of GITAM University for supporting research work.Figure. 1: Chemical structure of Melphalan and its impurities.a) Phthalimidoc)e)b) Ethyl esterElongatedd) MonolakylateMonohydroxyDOI: 10.9790/3008-1205016978f) Dihydroxywww.iosrjournals.org75 Page
Simaltaneous Determination Of Melphalan And Its Process-Related Impurities Using A Stabilityg)Dimerh) MelphalanFigure. 2: Typical HPLC chromatograms of melphalan and impurities under different stress conditions.a) Chromatogram of Limit of Quantification solutionb) Chromatograms of sample solutionc)Chromatogram of melphalan and impurities mixtureDOI: 10.9790/3008-1205016978www.iosrjournals.org76 Page
Simaltaneous Determination Of Melphalan And Its Process-Related Impurities Using A Stabilityd)Chromatogram of melphalan and impurities under acid stress conditione)Chromatogram of melphalan and impurities under base stress conditionf)Chromatogram of melphalan and impurities under oxidative stress conditiong)Chromatogram of melphalan and impurities under heat stress conditionDOI: 10.9790/3008-1205016978www.iosrjournals.org77 Page
Simaltaneous Determination Of Melphalan And Its Process-Related Impurities Using A [11][12][13][14][15][16][17][18][19]United state pharmacopeia, USP 37 NF (40).British pharmacopeia, BP 2015.Indian Pharmacopeia.S.C. Sweetman, Martindale: The Complete Drug Reference, Pharmaceutical Press; 34th edition, 566 (2005).T. Facon, J.Y. Mary, and C. Hulin, Lancet, 370 (9594), 1209 (2007).K. Kiran Kumar and R.Venkata Nadh. A validated RP-HPLC method for the estimation of melphalan in tablet dosage forms. 2011:4: 863-867.Bergel F and Stock J.A.J.Chem.Soc., 1954: 2409.F. Malecki, J.Sulkowska and W.Weiss-Granzinska. Reversed Phase High performance Liquid Chromatography analysis ofmelphalan in Pharmaceuticals. 19931: 38: 255.DailymedBeckett H and Stenlake JP. Practical pharmaceutical chemistry. 4th ed., Part 2, London: Athlone press; 1997, pp. 275-325.Subbarayan Shanmugavel and Venkatachalam Karthikeyan. Analytical method development and validation of layer by layermagnetic nanoparticles of Methotrexate and Melphalan. Volume 3, Issue 3:1221-1253.ICH Q2 (R1), Validation of analytical procedures: Text and methodology, 2005.ICH Q1 (R2), Stability testing of New Drug Substances and Products, 2000.ICH, Photo stability testing of new drug substances and products Q1B.ICH Guidelines on validation of analytical Procedures definitions and terminology.Drugstability principles and practices third edition, edited by Jens T, Carstensen Rhodes CT. 2000.Validation of compendial methods The United States Pharmacopeia.2016:42.Jens T, Carstensen, Rhodes CT, Drug stability principles and practices, Marcel Dekker, New York, 2000.ICH Stability Testing of New Drug Substances and Products Q1A (R2), International Conference on Harmonization, IFPMA,Geneva, 2003.IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS) is UGC approved Journalwith Sl. No. 5012, Journal no. 49063.JagadeswaraRao K. “Simaltaneous Determination of Melphalan and Its Process-RelatedImpurities Using a Stability-Indicating and Validated Reverse Phase Hplc Method in a ShortRun Time.” IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS), vol. 12, no. 5,2017, pp. 69–78.DOI: 10.9790/3008-1205016978www.iosrjournals.org78 Page
* Corresponding author: Prof. Rama Rao Malla Abstract: Degradation pathway of melphalan was validated by stability indicating reverse phase liquid chromatographic method. melphalan was subjected to stress condition using acid, base or by oxidation, and photolysis. Significant deg
ENVIRONMENTAL ENGINEERING LABORATORY – SYLLABUS Exp. No. Name of the Experiment 1. Determination of pH and Turbidity 2. Determination of Conductivity and Total Dissolved Solids (Organic and Inorganic) 3. Determination of Alkalinity/Acidity 4. Determination of Chlorine 5. Determination of Iron 6. Determination of Dissolved Oxygen 7.
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11.3 Determination of Fat content in Khoa 98 11.4 Determination of Starch in Khoa 99 11.5 Detection of sucrose in Khoa 99 12 TABLE (CREAMERY), AND DESHI BUTTER 101 12.1 Preparation of Sample of Butter 101 12.2 Determination of Moisture in Butter 101 12.3 Determination of Fat and
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